Gene therapy for ultrarare diseases: a geneticist’s perspective

Gene therapy has made considerable strides in recent years. More than 4000 protein-coding genes have been implicated in more than 6000 genetic diseases; next-generation sequencing has dramatically revolutionized the diagnosis of genetic diseases. Most genetic diseases are considered very rare or ultrarare, defined here as having fewer than 1:100,000 cases, but only one of the 12 approved gene therapies (excluding RNA therapies) targets an ultrarare disease. This article explores three gene supplementation therapy approaches suitable for various rare genetic diseases: lentiviral vector-modified autologous CD34+ hematopoietic stem cell transplantation, systemic delivery of adeno-associated virus (AAV) vectors to the liver, and local AAV delivery to the cerebrospinal fluid and brain. Together with RNA therapies, we propose a potential business model for these gene therapies.


Current success in gene therapy
Although initial attempts at human gene therapy were met with complications and failures, there has been an increase in the number of approved gene therapy products in recent years [3,4].Below are lists of USand/or EU-approved gene therapies, including RNA therapies, adapted and updated from "The state of cell and gene therapy in 2023" (Tables 1 and 2) [5].These lists exclude cancer gene therapies.

Approved gene therapies
Luxturna, approved in 2017 for Leber's congenital amaurosis (LCA), has been a remarkable success, restoring vision in a manner often described as miraculous that subretinal injection of a recombinant adeno-associated virus (AAV) delivering the normal copy of the human RPE65 cDNA led to reversal of blindness [6].AAV depends on a helper virus to complete its life cycle and does not cause any known human diseases, and AAV rarely integrates into the host genome [7].This breakthrough underscores the potential of gene therapy to treat previously untreatable diseases.The success of Luxturna encouraged advancements in the field of gene therapy.Following Luxturna, Zolgensma, utilizing an AAV9 virus capsid, was an even greater success for gene therapy [8,9].Spinal muscular atrophy (SMA) is an excruciating degenerative disease in which the most severely affected infants do not develop the ability to sit, and others progressively lose motor and respiratory functions.SMA is a relatively common genetic disease, with an incidence of approximately 1 in 10,000 people, creating a substantial market for Zolgensma.From 2019 to 2021, three gene therapies using genetically (lentiviral vector) modified autologous CD34 + hematopoietic stem cell transplantation were approved: Zynteglo for β-thalassemia [10], Skysona for adrenoleukodystrophy (ALD) [11,12], and Libmeldy for metachromatic leukodystrophy (MLD) [13].
β-Thalassemia and ALD can also be treated with allogeneic hematopoietic stem cell transplantation (HSCT), but gene therapy offers a viable alternative when a suitable donor is unavailable, and autologous transplantation presents a lower risk than allogeneic transplantation.
In 2022, Upstaza, rAAV2-hAADC, was approved for treating aromatic l-amino acid decarboxylase (AADC) deficiency, marking the first gene therapy targeting the brain directly.The same year, Roctavian and Hemgenix were approved for treating hemophilia A [14] and B [15], respectively.Gene therapy for hemophilia has achieved success comparable to that of Zolgensma, driven by the relatively high prevalence of hemophilia (hemophilia A affects 1 in 5,617 males, and hemophilia B affects 1 in 19,283 males).This success is particularly important considering the expensive and inconvenient nature of coagulation factor infusions.In 2023, four more gene therapies were approved: Vyjuvek for epidermolysis bullosa [16]; Lyfgenia, which uses a lentiviral vector encoding the antisickling hemoglobin HbA T87Q , for sickle cell anemia [17]; Casgevy, which employs CRISPR-Cas9, the first gene editing therapy, to target the BCL11A erythroid-specific enhancer [18], also for sickle cell anemia; and Elevidys, which is used for Duchenne muscular dystrophy (DMD) [19], a genetic muscular degenerative disease affecting approximately 1 in 5,000 males.

Approved RNA therapies
There are more approved RNA therapies than gene supplementation therapies (Table 2).Antisense oligonucleotides (ASOs) and small interfering RNAs (RNAi) are two widely used strategies for silencing gene expression [20].

Clinical trials
There are also clinical trials for new treatments.Liver transduction of AAV-G6PC increases the long-term efficacy of treatment for glycogen storage disease type Ia [23].Gene therapies with different strategies, including liver depot gene therapy, have been tested for the treatment of glycogen storage disease type II (Pompe disease) [24,25].More genetic diseases of the eyes are being investigated.Treatment for patients with ABCA4associated Stargardt disease is conducted with an equine infectious anemia virus-driven vector (EIAV-ABCA4) [26].Antisense oligonucleotides rescue aberrant splicing caused by an ultrarare ABCA4 variant in children with early-onset Stargardt disease [27].AAV5-NR2E3 may attenuate retinal degeneration caused by rhodopsin and other gene mutations in patients with retinitis pigmentosa [28].Moreover, a single administration of lipid nanoparticles loaded with gene editor mRNAs could inactivate the Pcsk9 gene to treat genetic and acquired hypercholesterolaemia [19], and currently, the heart-1 study is ongoing [29].

Rare diseases having intense drug developments
Rare diseases, such as DMD, have been approached by different ways [30,31].Around 80% of DMD mutations are potentially amenable to exon skipping.Eteplirsen (Exondys 51, Sarepta Pharmaceuticals) was the first exon-skipping pharmacologic treatment approved by the FDA in 2016 [32].Exon 53 skipping, golodirsen (Vyondys 53, Sarepta Pharmaceuticals), increases the proportion of eligible DMD patients by a few percent [33].The readthrough drug ataluren (Translarna) targets approximately 13% of DMD patients who have a nonsense mutation [34].Moreover, Elevidys, an AAV-based gene supplementation therapy, supplies a copy of microdystrophin cDNA that could benefit all DMD patients [35].
Another example is Huntington's disease (HD) which is caused by a pathological expansion of CAG repeat on the huntingtin gene.There are several ways to decrease the expression of the mutant Huntingtin protein [36,37].Huntingtin suppression with ASOs specific to HD mutation linked single-nucleotide polymorphisms restores cognitive function in a mouse model of HD [38].Mutant huntingtin lowering ASOs can be delivered to the brain through systemic administration using apolipoprotein A-I nanodisks [39].miRNA can also lower huntingtin levels and preserves striatal volume and cognitive function in a humanized mouse model of HD [40].An orally available, brain penetrant, small molecule lowers huntingtin levels by enhancing pseudoexon inclusion [41].Overexpression of sterol regulatory element-binding protein 2 (SREBP2) in HD mice activates the transcription of cholesterol biosynthesis pathway genes, clears mutant huntingtin aggregates, and attenuates behavioral deficits [42].

Few treatments for ultrarare genetic diseases
Among these diseases for which gene therapy has been approved, SMA, DMD, and hemophilia A have the highest incidence, occurring in at least 1 in 10,000 individuals.The incidence of LCA ranges from 1 in 33,000 to 80,000.AADC deficiency, with only 140 documented cases (Orphanet Report November 2023), stands out as the sole ultrarare disease in this context.ALD (1:20,000-50,000) and MLD (1:40,000-100,000) have higher incidences than AADC deficiency; however, only a subset of patients are eligible for gene therapy, which must be administered before symptoms manifest.ALD can also be treated with allogeneic HSCT, which might pose challenges for Bluebird Bio to profit from these two products.Moreover, substantially more resources are being dedicated to gene therapy for cancer than for genetic diseases, particularly chimeric antigen receptor (CAR) T-cell therapy [43].RNA therapy has also achieved several successes, but its high application potential lies in the ability of its production to be scaled up to treat a large number of patients.Consequently, developing gene therapy for ultrarare genetic diseases often faces difficulty in securing funding or resources.

Burden of ultrarare genetic disease
Given the vast number of genetic diseases, many geneticists routinely encounter a wide variety of conditions in practice.An experienced geneticist can typically identify hundreds of genetic diseases.For instance, mucopolysaccharidoses (MPSs) encompass types I, II, III, IV, VI, and VII.Mucolipidoses (MLs) include sialidosis, galactosialidosis, MLII, and MLIII.Glycolipidoses include Gaucher disease, Niemann-Pick A/B, Niemann-Pick C, GM1 and GM2 gangliosidosis, and Fabry disease.Glycogenosis encompasses glycogen storage disease types IA, IB, II (Pompe disease), and III.Neurotransmitter deficiency includes AADC deficiency, deficiency of tyrosine hydroxylase (TH), deficiency of 6-pyruvoyl-tetrahydropterin synthase (PTPS), and deficiency of GTP cyclohydrolase 1 (GCH1).Other metabolic diseases include urea cycle disorders, aminoacidopathies, and organic acidurias.Skeletal diseases include Ehlers-Danos syndrome (EDS), spondyloepiphyseal dysplasia (SED), and osteogenesis imperfectia (OI).Additional categories include congenital generalized lipodystrophy, diseases involving DNA repair defects, and various etiologies of early infantile epileptic encephalopathy (EIEE).Recently, we described the genetic etiology of 34 patients with skeletal diseases, identifying 16 genes involved in these conditions [44], with an average of two patients per disease.During my 30-year practice, I have seen all the diseases mentioned here.Regrettably, few specific treatments are available, and among them, AADC deficiency is the only ultrarare disease for which gene therapy has been developed [45].With sufficient resources, many of these conditions can be treated with gene therapy.
Gene therapy modalities suitable for treating ultrarare genetic disease (Fig. 1) Lentiviral vector-modified autologous CD34 + hematopoietic stem cell transplantation [46] (Fig. 2) Among the 12 previously mentioned approved gene therapies for genetic diseases, six utilize lentiviral vectormodified HSCT for patients with adenosine deaminase deficiency, β-thalassemia, MLD, ALD, and two distinct products for sickle cell anemia.Lentiviral vectors, such as those used to treat thalassemia, are capable of delivering complex tissue-specific expression cassettes to nondividing cells [10] without promoting leukemogenesis, in contrast to the γ-retroviral vectors used in earlier clinical studies [47].In theory, any genetic disease treatable by traditional allogeneic HSCT could also benefit from lentiviral vector-modified HSCT; moreover, autologous transplantation in gene therapy is considered safer than allogeneic transplantation.Allogeneic HSCT has proven effective for treating diseases such as Gaucher disease and MPSI, although enzyme replacement therapy (ERT) is preferred due to safety considerations.However, in diseases affecting the brain, such as Gaucher disease type III and MPSIH/S, allogeneic HSCT is still considered a treatment option [48].It is understood that transplanted hematopoietic stem cells can differentiate into phagocytic cells that migrate to the brain as microglia [49], which can then clear abnormal metabolites in diseases such as ALD or secrete molecules to rescue host brain cells in lysosomal storage diseases.
The advantages of lentiviral vector-modified HSCT over allogeneic HSCT also include the use of stronger promoters to increase product expression and codon optimization to increase translation efficiency.Genetic engineering can add a secretory leader peptide to the gene, achieving high blood levels of the transgene product akin to ERT.Lentiviral vector-modified HSCT can maintain high and stable blood levels of the expressed protein, unlike the pulsatile blood protein levels observed in ERT.Recently, brain-targeted ERT, developed by adding brain-penetrating epitopes to the infused protein [50], such as a transferrin epitope or an antibody to the receptor on the infused protein, has been shown to facilitate transferrin receptor-mediated transport across the blood-brain barrier.This strategy can be easily adapted to lentiviral vector-modified HSCT by modifying the cDNA sequence in the vector [51].
However, lentiviral vector-modified HSCT has limitations and complications.Currently, it is impossible to regulate the expression level of the transgene after viral Fig. 1 Application of gene therapy for ultrarare diseases.CGD chronic granulomatous disease, EDS Ehlers-Danos syndrome, EE epileptic encephalopathy, ML mucolipidoses, MPS mucopolysaccharidoses, NBIA neurodegeneration with brain iron accumulation, OA organic acidurias, OI osteogenesis imperfecta, SCID severe combined immunodeficiency, SED spondyloepiphyseal dysplasia, neurotransmitter deficiency, UCD urea cycle disorders vector transduction.While the overexpression of the transgene may be tolerable in conditions such as lysosomal storage diseases, where treatment necessitates the delivery of large amounts of enzyme systemically or to the cerebrospinal fluid (CSF), it can be toxic in other contexts.For instance, in gene therapy for AADC deficiency, the enzyme responsible for monoamine neurotransmitter production is delivered directly to the putamen to prevent ectopic or excessive dopamine production in the brain.Similarly, overexpression of β-globin in therapies for β-thalassemia could lead to a relative deficiency of α-globin, essentially converting the disease to α-thalassemia.Thus, the lentiviral vector used for gene therapy of β-thalassemia contains regulatory elements for the β-globin gene.While lentiviral vectors have been proven to be safer than retroviral vectors and are not associated with vector insertion-related leukemia, they have recently been suspected to cause myelodysplasia (MDS) [52].Given the novelty of gene therapy, a careful assessment of risks and benefits is necessary before proceeding with treatment.

Systemic delivery of AAV vector to the liver (Fig. 3)
Among the 12 approved gene therapies for genetic diseases, two target the liver (hemophilia A and B), one targets the nervous system (SMA), and one targets the muscle (DMD) using systemic delivery of AAV vectors.The systemic infusion of Zolgensma is used to treat children with SMA, but plans are in place to deliver the vector to the CSF in adult patients [53].Elevidys, recently approved for DMD treatment at a dose of 1.33 × 10 14 vector genomes per kilogram (vg/kg) of body weight, is still under evaluation for both efficacy and potential adverse effects.Systemic delivery of AAV to muscles requires high doses and is more likely to cause complications, including liver toxicity and microangiopathy [54].Conversely, since the liver retains more than 90% of systemically infused AAV vectors, systemic delivery to the liver requires only one-tenth the dose needed for muscle targeting, significantly reducing the risk of complications [55].Valoctocogene roxaparvovec (AAV5-hFVIII-SQ) contains a coagulation factor VIII cDNA driven by a liver-selective promoter [14].In a phase 3 study, 134 participants with hemophilia A received a single infusion of 6 × 10 13 vg/kg of vector.The mean factor VIII activity level at one year increased by 41.9 IU per deciliter.The mean annualized rates of factor VIII concentrate use and treated bleeding after week 4 decreased after infusion by 98.6% and 83.8%, respectively.There was no mortality in the trial.Etranacogene dezaparvovec is an AAV5 vector expressing the Padua factor IX variant.In a phase 3 study, 54 men with hemophilia B received 2 × 10 13 genome copies per kilogram of body weight of the vector.The annualized bleeding rate decreased from 4.19 during the lead-in period to 1.51 during months 7 through 18 after treatment [15].
In the case of hemophilia, the liver is pivotal because it produces coagulation factors.Many other genetic diseases, including metabolic diseases such as organic acidurias, urea cycle disorders, and aminoacidopathies, are also caused by liver dysfunction.Like all vector-mediated gene therapy, the efficacy can be enhanced with stronger or more appropriate promoters, codon optimization, secretory leader peptides, and epitopes targeting organs such as the brain.Systemic delivery of the AAV44.9-Mmutvector has been shown to prevent lethality and lower disease-related metabolites in methylmalonic acidemia mice.Tissue biodistribution and transgene expression studies in treated mice showed that AAV44.9 was efficient at transducing the liver and heart [56].Pompe disease is a lysosomal storage disorder causing skeletal muscle weakness and cardiomyopathy.Recent data reveal that 2 × 10 11 vg/kg of AAV2/8-LSPhGAA, containing a liver-specific promoter/enhancer and a leader sequence, transduced all hepatocytes which led to partial biochemical correction in adult GAA-KO mice with Pompe disease [57].
However, systemic AAV delivery is hindered by preexisting antibodies to the viral capsid, excluding some patients from therapy [58].Additionally, hepatocyte turnover can lead to a decrease in transgene expression over time, as in most liver-directed gene therapies, although the duration until therapeutic effects diminish Fig. 3 Mechanism of systemic AAV vector delivery to the liver.After intravenous infusion, the majority of AAV vectors are taken up by the liver.The transduced hepatocytes restore their functions, such as by conducting an enzyme reaction.If the transgene encodes a secretory protein, the protein can be released into the circulation to treat systemic diseases.The secretary protein, if it contains a brain-targeting epitope, can enter the brain remains uncertain [55,59].Last, although AAV is not an integrating virus, a small amount of integration into the host genome has been observed [60], and the long-term implications of this viral integration are yet to be fully understood.
Local delivery of AAV vectors to the CSF and brain for neurological disease treatment (Fig. 4) Targeting is still the greatest challenge in gene therapy.For example, scientists are working on the development of modified AAV vectors that target muscle cells while also reducing liver uptake [61].Reprogramming of the AAV capsid to mediate brain gene delivery has also been undertaken [62].However, AAV vectors can be easily administered directly into CSF spaces (intrathecal or intracisternal) or the brain parenchyma to treat diseases affecting the brain, spinal cord, eyes, and ears.CSF and brain delivery have several advantages.First, the vector quantity required is substantially less than that needed for systemic infusion-an order of magnitude less than systemic AAV delivery to the liver.This reduction saves costs and minimizes the risk of systemic complications such as liver damage.Second, the challenge posed by preexisting antibodies is less important for CSF and brain delivery due to the immune privilege of the central nervous system.Third, since neurons do not divide, the therapeutic effects of AAV can be long-lasting.Fourth, delivering the vector directly to the brain reduces the risk of ectopic expression of the transgene.
Among the 12 approved gene therapies for genetic diseases, one targets the eyes (LCA), and one targets the putamen (AADC deficiency) via local injection of AAV vectors.Both utilize AAV2, which exhibits high tropism for neuronal cells and limited tissue distribution.These treatments use minimal amounts of vector and are characterized by high efficacy and a lack of significant complications.Currently, gene therapy is advancing for several eye and ear diseases [63].Additionally, various brain disorders could benefit from brain-directed AAV injections.Monoamine neurotransmitter deficiencies can be treated by putamen injections [64].Neurodegeneration with brain iron accumulation (NBIA) could be treated by injection into the putamen or globus pallidus [65].Primary dystonia may be relieved by targeting the thalamus [66].The delivery of AAV vectors to CSF spaces is an option for treating diseases with more widespread brain pathologies.Intracerebroventricular AAV delivery in humans predominantly transduces ependymal cells [67].Delivery through lumbar puncture is both convenient and practical, particularly for spinal cord diseases [68].Direct injection into the cisterna magna (intracisternal injection) may provide a balanced vector distribution between the brain and spinal cord [68].These methods could benefit many brain diseases or systemic diseases Fig. 4 The mechanism of AAV vector delivery to the brain.AAV vectors can be administered directly into the brain parenchyma or injected into CSF spaces (e.g., by lumbar puncture) and the vectors then migrate to the spinal cord and brain.AAVs can transduce neural cells in the brain and ameliorate their dysfunction.Transgenes may be secreted to alleviate the dysfunction of other cells with brain manifestations, including some lysosomal storage diseases [69].
The primary limitation of brain delivery is whether the vector distribution is sufficiently broad to encompass all affected brain regions.For example, CSF delivery typically leads to the transduction of a small number of neurons in the putamen, while intraputaminal injections do not affect cells in the cortex or cerebellum.Moreover, since brain pathologies in most diseases are not reversible, the potential efficacy of gene therapy can be limited.

RNA therapy and gene editing for ultrarare disease
The abovementioned three modules of gene therapies are mostly involved in gene supplementation, but RNA therapy is also emerging as one of the most promising treatments for ultrarare diseases [2].Single-stranded DNA or RNA oligonucleotides bind RNA and block gene expression, modulate splicing, cleave DNA•RNA hybrids via RNase H, and target miRNAs [20].Doublestranded short interfering RNAs (siRNAs) bind the protein machinery of the RNA-induced silencing complex (RISC), and the RISC directs its bound small RNA to target complementary RNAs and represses their expression through mRNA cleavage, degradation, and translational repression [70].Modification of DNA or RNA oligonucleotides by phosphorothioate (PS) linkages and chemical modifications greatly improves their stability, binding to serum proteins, and binding affinity for their complementary sequences [71,72].
ASOs and RNAi can reduce mRNA levels and suppress the expression of mutated toxic gene products, as observed in HD [37], or target upstream or downstream genes in metabolic pathways to mitigate the detrimental effects of mutations, such as acute hepatic porphyria [73].Additionally, ASOs can regulate mRNA splicing; for instance, in treating DMD, they can splice out exons containing missense or nonsense mutations to restore protein translation, producing a shorter but functional dystrophin protein that alleviates symptoms [30].In more unique cases, ASOs interfere with the binding of splice suppressors, as in the treatment of SMA, to enable correct splicing [74].
Although RNA therapy drugs require regular administration to treat genetic diseases, oligonucleotides can be produced and purified like small molecule drugs, offering the same advantages of low production costs and scalability.The behaviors of ASOs are quite consistent, resulting in predictable therapeutic doses, routes of administration, frequencies of dosing, and potential side effects [75].Recently, n-Lorem collaborated with Ionis Pharmaceuticals to discover and develop personalized ASOs for one patient at a time-N-of-1 therapies [76,77].The possibilities of ASOs and RNAi therapies for treating monogenic disorders have recently been reviewed [78].
Gene editing, mainly using CRISPR/Cas, represents another powerful tool for effectively disrupting gene expression [79].Currently, the first approved clinical application of CRISPR/Cas9 is for treating sickle cell anemia.Due to concerns about the risks associated with double-strand breaks [18], base editing, a derivative of CRISPR/Cas9 that modifies bases without inducing double-strand breaks, is a safer alternative that has catalyzed several clinical trials [80].Nonetheless, the potential for off-target effects [81] necessitates cautious application of base editing in treatments for ultrarare diseases.In Table 3, we compare the treatment modules mentioned in this article and note the feasibility of applying them to treat ultrarare diseases.

Funding, reimbursement, and business model
Although gene therapy has the potential to transform the lives of people living with these devastating rare diseases, accessing these new therapies is far from straightforward for patients [82].The obstacles in developing treatments for rare diseases extend beyond technological issues, including funding, reimbursement strategies, and business models.While most currently approved gene therapies target rare diseases, they collectively impose a substantial financial impact on health and insurance systems [83].This has prompted the proposal of various payment methods and policies to ensure that patients can access the benefits of gene therapy, for example, to increase drug affordability through health care loans [84].A business model for the treatment of ultrarare diseases is also difficult.One innovative approach suggested by the n-Lorem organization, particularly for ultrarare diseases affecting fewer than 30 people globally, involves treating one patient at a time using a nonprofit model [76].
Nevertheless, the gene therapy modalities discussed in this article-lentiviral vector-modified autologous CD34 + hematopoietic stem cell transplantation, systemic delivery of AAV to the liver, delivery of AAV to the CSF and brain, and RNA therapies-have the potential to treat multiple genetic diseases.In a regulatory environment conducive to innovation, a company could specialize in a single technique for multiple diseases, thereby saving on development costs, vector production, biodistribution, and toxicity testing.By partnering with patient groups and medical societies eager to deliver treatments to their patients, clinical trials could be designed more cost-effectively [85].For example, when we developed Upstaza for AADC deficiency, the first and only braindirected gene therapy targeting an ultrarare disease, we developed it through academic research grants before transitioning to a commercial setting [86].These strategic approaches will allow companies to remain profitable while expanding access to treatments for more patients with ultrarare diseases.

Conclusions
This article overviews current successes in gene therapy, including autologous lentiviral vector-modified hematopoietic stem cell transplantation to treat hematological and neurological diseases and AAV vector-mediated gene therapy to treat eye, liver, and neurological diseases.These new technologies provide hope for the thousands of individuals with rare genetic diseases.However, both the high risk and cost of gene therapy prevent its rapid development, especially for ultrarare diseases with only a small number of eligible patients.In this article, we propose several gene therapy technologies that are suitable for treating rare genetic diseases.For example, in the systemic delivery of AAV vectors, liver targeting requires fewer vectors but can treat both liver and systemic diseases.Direct delivery of the AAV vector to the nervous system can also treat neurotransmitter deficiency, primary dystonia, and NBIA.A company can therefore specialize in one technology that can target multiple ultrarare diseases to decrease the financial burden of gene therapy development.We hope that more patients with ultrarare genetic disease can receive gene therapy soon.

Fig. 2
Fig. 2 Mechanisms of lentiviral vector-modified autologous CD34 + hematopoietic stem cell transplantation.Transduction of hematopoietic stem cells can correct functional defects in blood cells, including T cells, B cells, and macrophages.Transgenes can encode secretory proteins that are secreted into the systemic circulation.The secreted protein, if it contains a brain-targeting epitope, can enter the brain.Bone marrow-derived cells can also migrate to the brain and differentiate into microglia, which can ameliorate brain defects

Table 1
List of US-and/or EU-approved gene therapies

Table 2
List of US-and/or EU-approved RNA therapies